ABSTRACT Gag is capable of self-assembly in vitro, although it is generally accepted that host factors play a decisive role for immature Gag trafficking. Although many of the cellular proteins and mechanisms co-opted for viral budding and release are well understood, the full identity and the role of cellular proteins participating in immature Gag assembly remains incomplete. This Project focuses on identifying the composition of cellular factors interacting with Gag/Gag-Pol throughout the Gag assembly pathway contributing to both the dynamic assembly and cellular trafficking of Gag. To provide fundamental information on Gag assembly, early steps will be interrogated in vitro at the single molecule level and later in cells. Computational methodologies will also integrate the structural and biophysical findings into coherent structural models of higher-order assemblies. Importantly, the structure of the first retroviral polyprotein, the protease-reverse transcriptase fusion protein of prototype foamy virus has been solved. This sets the stage for additional and important structural characterization of more complex retroviral polyproteins, which is required for understanding how cellular host factor interactions contribute to Gag and Gag-Pol assembly. The proposed investigations will strongly leverage the complementary scientific expertise within and collaborative nature of the HIVE Center and take advantage of cutting-edge technologies available to address the following Specific Aims: Aim 1. Pulse-labeling and quantitative mass spectrometry to characterize Gag and Gag-Pol, assembly intermediates including associated proteins, and RNA, with subsequent functional analysis (collaborators are: Williamson, Lyumkis, Marcotrigiano, Arnold, Griffin, Torbett); Aim 2. Single- molecule analysis of Gag assembly in vitro and in vivo to gain insight into specific RNA contributions and Gag oligomerization required for Gag assembly (collaborators are: Millar, Musier-Forsyth, Torbett, Williamson); Aim 3. Structural studies of Gag, Gag-Pol and Pol of HIV-1 and PFV (collaborators are: Arnold, Marcotrigiano, Lyumkis, Griffin, Torbett, Williamson); and Aim 4. Mesoscale modeling of Gag during assembly (collaborators are: Olson, Levy, Goodsell). Understanding the temporal interplay of cellular factors contributing to immature Gag assembly and progression, as well as the strategic role of Gag-Pol, and elucidating the structural basis for these interactions will provide a more comprehensive understanding of an area in retroviral biology that is currently not well understood. Elucidating the structural basis of identified cellular factors that interact with immature Gag and Gag-Pol will allow development of novel small chemical probes for further interrogation of these interactions.